Major histocompatibility complex (MHC) class II antigens are important in vertebrate immune system, which present peptides to CD4(+) T cells. In the present study, cDNAs encoding MHC class II B gene were isolated from the cDNA library of half-smooth tongue sole (Cynoglossus semilaevis), and the full length cDNA sequences were got by rapid amplification of cDNA ends polymerase chain reaction. The polymorphism of its open reading frame, 3' untranslated region and intron 1 was studied. Nineteen class II B alleles were identified from nine individuals and clustered into two groups, designated as Cyse-DAB and Cyse-DBB. The deduced amino acid sequences among Cyse-DAB and Cyse-DBB alleles shared identities from 94.0% to 99.6% and 92.4% to 99.6%, respectively, while the identities between Cyse-DAB and Cyse-DBB genes varied from 85.1% to 92.0%. Three Cyse-DAB alleles and one Cyse-DBB allele were observed in each of two individuals, and three Cyse-DBB alleles and one Cyse-DAB allele in another individual, which indicated at least two loci existed in each gene. Two different 3' UTR sequences were identified and one belonged to Cyse-DAB, the other belonged to Cyse-DBB. Both five Cyse-DAB and five Cyse-DBB intron 1 sequences were identified from genomic sequences, among which two sequences of each gene were identified in a single individual, which suggested the existence of at least two Cyse-DAB and two Cyse-DBB loci. Both the two genes'-specific tissue and developmental stage expression were studied by reverse-transcription polymerase chain reaction, which showed that the two genes had similar expression patterns in tissue study with high expression in spleen and kidney, low expression in liver, gill and ovary, moderate expression in brain, heart, intestine and testis. While in developmental analysis, Cyse-DBB had higher expression than Cyse-DAB in early developmental stages, which indicated that the two genes might have different functions in those stages. Therefore, in half-smooth tongue sole, two different MHC class II B genes exist and could differentiate from each other whether by sequence analysis or by developmental stage expression study.